Abstract
As an adaptation to life in a world with predictable daily changes, most eukaryotes and some prokaryotes have endogenous circadian (approximately 24 h) clocks. In plants, the circadian clock regulates a diverse range of cellular and physiological events from gene expression and protein phosphorylation to cellular calcium oscillations, hypocotyl growth, leaf movements, and photoperiod-dependent flowering. In Arabidopsis (Arabidopsis thaliana), as in other model organisms, such as Drosophila (Drosophila melanogaster) and mice, circadian rhythms are generated by molecular oscillators that consist of interlocking feedback loops involving a number of elements. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYLS (LHY) are closely related single myb transcription factors that have been identified as key elements in the Arabidopsis oscillator. Research in other model organisms has shown that posttranslational regulation of oscillator components plays a critical role in the generation of the approximately 24-h cycles. To examine the role of posttranslational regulation of CCA1 and LHY in the Arabidopsis oscillator, we generated transgenic plants with tagged CCA1 and LHY under the control of their own promoters. We have shown that these tagged proteins are functional and can restore normal circadian rhythms to CCA1- and LHY-null plants. Using the tagged proteins, we demonstrate that CCA1 can form both homodimers and heterodimers with LHY. Furthermore, we also show that CCA1 is localized to the nucleus in vivo and that there is no significant delay between the translation of CCA1 and its translocation to the nucleus. We discuss our findings in the context of the functioning of the Arabidopsis oscillator.